Transgene containment via maternal inheritance and male sterility in gm crops by henry daniell
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Transgene containment via maternal inheritance and male sterility in gm crops by henry daniell

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Transgene containment via maternal inheritance and male sterility in GM crops by Henry Daniell...

Transgene containment via maternal inheritance and male sterility in GM crops by Henry Daniell

Summary
The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant
species has been recognized as a potential environmental concern. Until the environmental impact of
novel genes on indigenous crops and weeds is thoroughly investigated, practical and regulatory
considerations might require the adoption of gene containment approaches for future generations of GM
crops. To date, most molecular approaches with potential for controlling gene flow among crops and
weeds have focused on maternal inheritance, male sterility, and seed sterility.This presentation will focus
on the use of maternal inheritance and cytoplasmic male sterility for transgene containment. Because no
single strategy will be broadly applicable to all crop species, a combination of more than one approach
might prove most effective for engineering failsafe mechanisms for the next generation of GM crops.
Concerns about the environmental impact of GM crops currently limit their widespread acceptance
around the world. Many of these concerns focus on the premise that such transfer could potentially result
in the emergence of “superweeds” resistant to herbicides or the introduction of undesired traits into
related crop plants. Gene flow depends upon several factors, including the specific crop, its location, the
presence of out-crossing wild relatives/sexually compatible crops, the competitive nature (advantages
and disadvantages) of the introduced trait, and the environmental consequences of neutral traits. Two
mechanisms are responsible for the movement of genes among crops and their wild relatives/related
crops: dispersal in viable pollen, or dissemination in seed (that later germinates and produces viable
pollen). This presentation will focus on the dispersal via pollen. The potential for gene flow via pollen
depends on several factors, including the amount of pollen produced, longevity of pollen, dispersal of
pollen (via wind, animal), plant/weed density, dormancy/rehydration of pollen, survival of pollen from
toxic substances secreted by pollinators, the distance between crops and weeds, and whether these plants
are sexually receptive to the crop.

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  • 1. Transgene containment via maternal inheritance and male sterility in GM crops Henry DaniellUniversity of Central Florida, Dept. Molecular Biology & Microbiology, Biomolecular Science,Building #20, Room 336, Orlando FL 32816-2364, U.S.A.SummaryThe potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plantspecies has been recognized as a potential environmental concern. Until the environmental impact ofnovel genes on indigenous crops and weeds is thoroughly investigated, practical and regulatoryconsiderations might require the adoption of gene containment approaches for future generations of GMcrops. To date, most molecular approaches with potential for controlling gene flow among crops andweeds have focused on maternal inheritance, male sterility, and seed sterility.This presentation will focuson the use of maternal inheritance and cytoplasmic male sterility for transgene containment. Because nosingle strategy will be broadly applicable to all crop species, a combination of more than one approachmight prove most effective for engineering failsafe mechanisms for the next generation of GM crops.Concerns about the environmental impact of GM crops currently limit their widespread acceptancearound the world. Many of these concerns focus on the premise that such transfer could potentially resultin the emergence of “superweeds” resistant to herbicides or the introduction of undesired traits intorelated crop plants. Gene flow depends upon several factors, including the specific crop, its location, thepresence of out-crossing wild relatives/sexually compatible crops, the competitive nature (advantagesand disadvantages) of the introduced trait, and the environmental consequences of neutral traits. Twomechanisms are responsible for the movement of genes among crops and their wild relatives/relatedcrops: dispersal in viable pollen, or dissemination in seed (that later germinates and produces viablepollen). This presentation will focus on the dispersal via pollen. The potential for gene flow via pollendepends on several factors, including the amount of pollen produced, longevity of pollen, dispersal ofpollen (via wind, animal), plant/weed density, dormancy/rehydration of pollen, survival of pollen fromtoxic substances secreted by pollinators, the distance between crops and weeds, and whether these plantsare sexually receptive to the crop.Following pollination and reproduction, dispersal of seeds from GM plants may also occur among weedyrelatives during harvest, transportation, planting, and reharvest, giving rise to mixed populations. If theseGM seeds germinate, grow, and reproduce, there is a risk that interbreeding with a sexually compatibleweedy species could produce a fertile hybrid. Further crossing with the weed species (introgressivehybridization) could then result in new weeds that have acquired the GM trait. This again depends onthe persistence of the crop among weeds and probability of forming mixed stands. • 149 •
  • 2. Maternal inheritanceThree modes of plastid genome inheritance have been described in the literature. Uniparentalmaternal plastid inheritance is observed in a majority of angiosperms [1]. This was first describedalmost a hundred years ago for Mirabilis jalapa. Uniparental maternal inheritance is achieved throughplastid exclusion from the generative cell during the first haploid pollen mitosis; all plastids aredistributed into the vegetative cell and the generative cell is free of plastids. Therefore, the spermcells formed from the generative cell are free of plastids [1]. If the generative cell acquires a fewplastids, they degenerate during maturation and the sperm cell becomes free of plastids [1]. Incereals, both generative and sperm cells contain plastids but they are removed from the spermnucleus before or during the process of fertilization. However, rare exceptions to uniparentalmaternal inheritance have been reported. For example, when 97,613 Antirrhinum majus plants weretested, one plant among 4,066 showed paternal plastids [2]. Among 787,329 hybrid Setaria italicaplants tested, 241 paternal transmissions of atrazine resistance were observed [2]. Occasionaltransmission of paternal plastids in tobacco has been reported [3].In a few exceptions among angiosperms, such as Oenothera or Medicago [4], biparental plastidinheritance has been reported. This is caused by equal distribution of plastids during the firsthaploid pollen mitosis into generative and vegetative cells. Therefore, the sperm cells transmitplastids into egg cells. Extraordinarily, uniparental paternal inheritance of plastids has beenreported in the kiwi plant [5]. Aforementioned exceptions demonstrate the need to develop alternateapproaches to eliminate rare paternal or biparental transmission of transgenes engineered via thechloroplast genome.Maternal inheritance of transgenes and prevention of gene flow via pollen in chloroplast transgenicplants have been successfully demonstrated in several plant species, including tobacco [6,7], tomato[8], cotton [9], soybean [10] and carrot [11]. Unlike many other containment strategies, the maternalinheritance approach has already been tested in the field. Scott and Wilkinson [12] studied plastidinheritance in natural hybrids collected from two wild populations growing next to oilseed rapealong 34 km of the Thames River in the UK and assessed the persistence of 18 feral oil seed rapepopulations over a period of three years. They analyzed several factors that would influence themovement of chloroplast genes from crops to wild relatives, including the mode of inheritance ofplastids and incidence of sympatry (the occurrence of species together in the same area) to quantifyopportunities for forming mixed populations and persistence of crops outside agriculture limits forintrogression. Despite some (0.6–0.7%) sympatry between the crop and weed species, mixed standsshowed a strong tendency toward rapid decline in plant number, seed return, and ultimatelyextinction within three years. Thus, they concluded that gene flow should be rare if plants aregenetically engineered via the chloroplast genome.In addition to maternal inheritance, chloroplasts genetic engineering approach offers a number ofadvantages, including high-level transgene expression, multi-gene engineering in a singletransformation event, lack of gene silencing, position effect and pleiotropic effects [13,14]. Thus,maternal inheritance of chloroplast genomes is a promising option for gene containment.Chloroplast genetic engineering has now been shown to confer resistance to herbicides [6], insects • 150 •
  • 3. [18,19] , disease [20], salt [11] and drought [21], as well as to produce antibodies, biopharmaceuticals,and vaccine antigens [15-17]. Several reports from the European Environment Agencies haverecommended chloroplast genetic engineering as a gene containment approach.Male sterility systemsMale-sterility-inducing cytoplasms are known for over a century. Cytoplasmic male sterile inbred lineshave been widely used in hybrid seed production of many crops. The first application of cytoplasmicmale sterility is for hybrid seed production, a major contribution towards the “Green Revolution”. Theuse of cytoplasmic male sterility in hybrid seed production has been recently reviewed by Havey (22).The use of CMS for hybrid seed production received a “black eye”after the epidemic of Bipolaris maydison T-cytoplasmic maize. This epidemic is often cited as a classic example of genetic vulnerability of ourmajor crop plants. In addition to Southern corn blight (CMS-T), cold susceptibility (CMS Ogura) andSorghum Ergot infection in the unfertilized stigma have been reported. But these disease linkages weresuccessfully broken by somatic cell genetics and conventional plant breeding (22).Hybrids of other crop plants may be produced using nuclear male sterility. A natural source of nuclearmale sterility was identified in leek (23). Engineered sources of nuclear male sterility have beendeveloped in model systems (24). GM rapeseed containing the Barstar Barnase male sterility systemcomprises ~10% of the commercially cultivated crop in Canada and is one of the few GMOs cleared foragricultural use in Europe. One problem with these nuclear transformants is that they segregate formale fertility or sterility and must be over planted and rogued by hand or sprayed with herbicides toremove male-fertile plants.Major investments of time and resources are required to backcross a male-sterility-inducing cytoplasminto elite lines. These generations of backcrossing could be avoided by transformation of an organellargenome of the elite male-fertile inbred to produce female inbred lines for hybrid seed production (22).Because the male-fertile parental and male-sterile transformed lines would be developed from the sameinbred line, they should be highly uniform and possess the same nuclear genotype, excluding mutationsand residual heterozygosity (22). Therefore, the male-fertile parental line becomes the maintainer lineto seed-propagate the newly transformed male-sterile line (22). A few generations of seed increaseswould produce a CMS-maintainer pair for hybrid seed production. An additional advantage oforganellar transformation would be the diversification of CMS sources used in commercial hybrid-seedproduction. Transformation of the chloroplast genome would allow breeders to introduce differentmale-sterility-inducing factors into superior inbred lines. Introduction of a male-sterility inducingtransgene into one of the organellar genomes of a higher plant would be a major breakthrough in theproduction of male-sterile inbred lines (22). This technique would be of great potential importance inthe production of hybrid crops by avoiding generations of backcrossing, an approach especiallyadvantageous for crop plants with longer generation times (22). Moreover, transgenes that areengineered into our annual crops could be introgressed into wild crops, persist in the environment andhave negative ecological consequences. Therefore, it may be necessary to engineer a male sterilitysystem that is 100% effective (22). Such a cytoplasmic male sterility system engineered via thechloroplast genome will be presented. • 151 •
  • 4. ConclusionsThere is currently a paucity of data on the environmental impact of specific GM traits. Nevertheless, it islikely that for the near future, regulatory restrictions are likely to dictate that gene-containment systemswill have to be developed for future GM crop releases. At present, no effective gene containment methodis available for all GM crops, and considerable investment and research is needed to develop thetechnologies outlined above.It is clear that the characteristics of seed and pollen production, dispersal, and potential outcrossing mustbe determined for each specific crop in each specific environment. Different crop species have differentrates of autogamy and outcrossing, and some crops have hybridizing wild relatives only in certaingeographical locations. It will also be important to allay concerns that crops engineered with alteredpollination, flowering, or male sterility patterns for the purpose of gene containment will not impact thewider biodiversity of insects, bird and wildlife in existing ecosystems.As shown above, both biological containment measures have been developed to control gene flow throughpollen or seed. Male sterility has been already commercially exploited in Canola. It is very effective atpreventing out-crossing from GM crop to weeds or related non-GM crops. However, seeds produced fromnuclear male sterile GM crops by cross-pollination from weeds, may be a serious concern because seedsof such hybrids will produce fertile pollen that would carry the GM trait. Also, pollen is not produced in acrop that makes the seed, making it less desirable for the farmer because it would require cross-pollinationfrom a non-GM crop or must be propagated by artificial seed. Reversible male sterile systems engineeredvia the chloroplast genome should address these concerns. Maternal inheritance is a promising approachfor transgene containment with added advantages of high levels of transgene expression, rapid multigeneengineering, lack of position effect, gene silencing and pleiotropic effects. Currently, chloroplast geneticengineering has been enabled in tobacco, a non-food/feed crop as a bioreactor for production ofbiopharmaceuticals, monoclonals or biopolymers or to confer desired plant traits. It has been enabled inseveral major GM crops, including cotton and soybean. Chloroplast transgenic carrot plants withstand saltconcentrations that only halophytes could tolerate. Extension of chloroplast genetic engineeringtechnology to other useful crops will depend on the availability of the plastid genome sequences and theability to regenerate transgenic events.References1. Hagemann, R. (2004) Molecular Biology and Biotechnology of Plant Organelles. Eds. Daniell, H and Chase C., Kluwer Academic Publisher, The Netherlands, 2004, Chap. 4, pp 87-108.2. Wang, T., et al. (2004) Theor. Appl. Genet. 108: 315-20 (2004).3. Medgyesy, P., Pay, A. & Marton, L. (1986) Molecular and General Genetics. 204, 195-198.4. Smith, S.E., Bingham, E.T. & Fulton, R.W. (1986) J. Heredity. 77, 35-38. • 152 •
  • 5. 5. Cipriani, G., Testolin, R. & Morgante (1995) Mol. Gen. Genet. 247, 693-697.6. Daniell, H., Datta, R., Varma, S., Gray, S., & Lee, S.B. (1998) Nature Biotechnol. 16, 345–348.7. Daniell, H (2002) Nature Biotechnology 20, 581-586.8. Ruf, S., Hermann, M., Berger, I.J., Carrer,H, & Bock, R. (2001) Nature Biotechnol. 19, 870–875.9. Kumar, S., Dhingra, A. & Daniell, H. (2004) Plant Mol. Biol. in press.10. Dufourmantel, N. et al. (2004) Plant Mol. Biol. in press.11. Kumar, S., Dhingra, A. & Daniell, H. (2004) Plant Physiol. in press12. Scott, S.E. & Wilkenson, M.J. (1999) Nat. Biotechnol. 17, 390-392.13. Daniell, H., Khan, M. & Allison, L. (2002) Trends Plant Sci. 7, 84-91.14. Daniell, H. & Dhingra, A. (2002) Curr. Opin. Biotechnol. 13, 136-141 (2002).15. Daniell, H. et al. (2004) Molecular biology and biotechnology of plant organelles (Daniell, H. and Chase, C., eds), pp. 423-468. Kluwer Academic Publishers.16. Chebolu, S. & Daniell, H. (2004) Current Trends in Microbiology and Immunology, in press.17. Daniell, H., Carmona-Sanchez, O. & Burns, B.B. (2004) Molecular Farming (Fischer, R. and Schillberg, S., eds), pp.113-133, WILEY-VCH Verlag18. DeCosa, B., Moar, W., Lee, S. B., Miller, M., & Daniell, H. (2001) Nature Biotechnology 19, 71¯74.19. Kota, M., Daniell, H., Varma, S. Garczynski, S.F., Gould, F., & William, M. J. (1999) Proc. Natl. Acad. Sci. USA, 96, 1840¯1845.20. DeGray, G., Rajasekaran, K., Smith, F., Sanford, J., & Daniell, H. (2001) Plant Physiology 127, 852-862.21. Lee, S. B., Kwon, H. B., Kwon, S. J., Park, S. C., Jeong, M. J., Han, S. E., & Daniell, H. (2003) Mol. Breeding, 11, 1-13.22. Havey, M.J. (2004) In Molecular Biology and Biotechnology of Plant Organelles, eds. Daniell, H. & Chase, C.D. (Kluwer Academic Publishers, The Netherlands), pp 617-628.23. Smith, B., & Crowther, T. (1995) Euphytic 86, 87-94.24. Marian,i C., De Beuckeleer, M.,Truettner, J., Leemans, J.,& Goldberg, R.B. (1990) Nature 347, 737-741. • 153 •